The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
The electrical potential difference across a neuronal cell membrane is the result of an inequitable distribution of ions on either side of the membrane. In its resting state, a neuron has a high internal store of potassium ions (K+) with sodium ions (Na+) accumulated on the outside of the membrane. In such a state, the flow of ions across a membrane through non-gated channels is such that their movement causes no net change in charge. However, a perturbation of this resting flow results in an alteration of the membrane's potential.
Sodium channels are aqueous pores in the cellular membrane that regulate and provide a selective passage for sodium ions between the internal and external environments of a cell. Voltage-gated sodium (Nav) channels, i.e. those opened by changes in membrane potential, are largely responsible for the depolarization of the cell. When closed, they help maintain the neuron's resting potential, and when open, allow sodium ions to flow down the electrochemical gradient and depolarize the cell.
Nav channels are formed by proteins embedded within the cell's membrane and are typically complexes of a large glycoprotein called the α-subunit, which forms the channel's pore, and auxiliary β-subunits, which regulate the function of the α-subunit. γ- and δ-Subunits may also exist to regulate the α-subunit.
The α-subunit has four repeats, labelled I through IV, of the same 150 amino acid sequence. Each repeat contains six membrane-spanning regions labelled S1 through S6. The highly conserved S4 region, thought to be part of the channel that acts as its voltage sensor, has a positive amino acid at every third position, with hydrophobic residues between these. It is thought that when stimulated by a change in transmembrane voltage, this subunit moves from within the pore toward the extracellular side of the cell, allowing the channel to become permeable to ions that would otherwise have been blocked by the subunit's positive charges. This is sometimes referred to as the “activation gate”. Another “plug” located on the internal side of the channel is known as the “inactivation gate”.
The inner pore of Nav channels contains a selectivity filter made of negatively charged amino acid residues (aspartic acid and glutamic acid), which attract the positive Na+ ion and keep out negatively charged ions such as chloride. The mouth of the pore is some 1.2 nm wide, narrowing to about 0.3 by 0.5 nm wide, which is just large enough to allow a single Na+ ion with a water molecule associated to pass through whilst being small enough to exclude larger K+ ions. Different sized ions also cannot interact as well with the negatively charged glutamic acid residues that line the pore.
Nav channels exist in three conformational states: resting (closed), activated (open), and inactivated (closed).
In their resting state Nav channels are blocked on their extracellular side by the activation gate, the inactivation gate is open and the inside of the neuron is negatively charged relative to the outside. This difference in membrane potential is referred to as the resting potential (−70 mV).
In the activated state, opening of Nav channels in response to an electrical stimulus results in a rapid influx of sodium ions, causing more Nav channels to open and the cell becomes more positively charged (depolarized). In healthy neurons, the activated state is unstable and will rapidly transition to the inactivated state.
The inactivated state is achieved shortly after the sodium channel has been activated, when the internal inactivation gate then blocks the inside of the Nav channel while an outflow of potassium ions through voltage-gated potassium channels restores the membrane potential to its resting value. Although the external activation gate is open, sodium ions cannot flow through into the cell. Rapid inactivation is crucial for the normal activity of the cell.
Finally, the inactivation gate opens and the activation gate closes bringing the Nav channel back to its resting state. This places the channels in readiness to be activated again during the next action potential.
This process of transition between the conformational states (referred to as gating) occurs in the space of 2-3 milliseconds and in this manner, changes in membrane potential are propagated along the membrane from the point of stimulation. A self-propagating wave of depolarization down the axon of a neuron is known as an action potential. The more Nav channels that exist in a neuron's membrane, the faster the action potential will propagate down the axon. When it reaches the end of the axon, the action potential may electrically stimulate the membrane of an adjacent cell or release neurotransmitters into the synaptic cleft, which chemically open gated channels in the adjacent cell membrane. It is the rapid cycling through the resting, activated and inactivated states that allow them to sustain rapid trains of action potential. Thus, Nav channels are critical for the initiation and propagation of action potentials in neurons and, ultimately, the electrical activity of the central and peripheral nervous systems.
Nine distinct α-subunits of voltage gated sodium channels have been identified and their corresponding channels are known as Nav1.1-Nav1.9. These subtypes share more than 50% amino acid identity within the membrane-spanning domains and extracellular loops, and can be distinguished not only by differences in their sequence but also by their kinetics and expression profiles, as well as tissue localization. The tissue localizations of the nine isoforms vary greatly. Nav 1.4 is the primary sodium channel of skeletal muscle, and Nav 1.5 is primary sodium channel of cardiac myocytes. Navs 1.7, 1.8 and 1.9 are primarily localized to the peripheral nervous system, while Navs 1.1, 1.2, 1.3, and 1.6 are channels found in the central or central and peripheral nervous systems.
Notwithstanding the essential role of voltage-gated sodium channels in the central and peripheral nervous systems, it is now well established that they are also implicated in the aetiology and/or symptoms of various neuronal diseases and disorders (neuropathies). Perturbations of the normal gating cycle can lead to hyperexcitability (excessive firing of the cell), which is implicated in a number of neuropathies such as migraine, epilepsy, neuropathic pain and neurodegenerative diseases (Eijkelkamp N., et al, Brain, 2012, 135, 2585-2512)
Depending on the particular nerves involved, the neuropathy can be classified as a central or peripheral neuropathy. Central neuropathies arise from spinal cord, brainstem, thalamic, and cerebral damage or disease, while peripheral neuropathies arise from damage or disease of peripheral nerves.
The peripheral nervous system transmits information from the brain and spinal cord to every other part of the body. More than 100 types of peripheral neuropathy have been identified, each with its own characteristic set of symptoms, pattern of development, and prognosis. Impaired function and symptoms depend on the type of nerves—motor, sensory, or autonomic—that are damaged. Some people may experience temporary numbness, tingling, and pricking sensations, sensitivity to touch, or muscle weakness. Others may suffer more extreme symptoms, including burning pain (especially at night), muscle wasting, paralysis, or organ or gland dysfunction. Peripheral neuropathy may be either inherited or acquired. Causes of acquired peripheral neuropathy include systemic diseases (e.g. diabetes), physical injury (trauma) to a nerve, tumors, toxins, autoimmune responses, viral and bacterial infections, nutritional deficiencies, alcoholism, and vascular and metabolic disorders. Inherited forms of peripheral neuropathy are caused by genetic mutations.
Central neuropathy, as the name implies, is the result of damage to the central nervous system, i.e. brain and spinal cord. As with peripheral neuropathies, the causes are varied and include physical injury, disease and autoimmune responses.
A particular example of such a neuropathy is multiple sclerosis (MS), which is a chronic, often disabling, disease that randomly attacks the central nervous system. The progress, severity and specific symptoms of the disease cannot be predicted; symptoms may range from tingling and numbness to paralysis and blindness. MS is a devastating disease because people live with its unpredictable physical and emotional effects for the rest of their lives. Symptoms of MS are unpredictable and vary greatly from person to person and from time to time in the same person. They may include: fatigue, impaired vision, loss of balance and muscle coordination, slurred speech, tremors, stiffness, bladder and bowel problems, difficulty walking, short-term memory loss, mood swings and, in severe cases, partial or complete paralysis.
A significant contributor to non-remitting deficits in demyelinating neuroinflammatory diseases such as MS and the related Guillain-Barre's syndrome (GBS), and their respective animal models, experimental allergic encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN), is axonal loss. Recent studies have demonstrated that persistently activated sodium channels can trigger axonal injury by providing a sustained sodium influx that can drive reverse sodium-calcium exchange and sodium channel blockade can prevent axonal degeneration within white matter tracts in a variety of disease models. In addition, it has been demonstrated that the sodium channel blocker phenytoin inhibits immune cells in the neuroinflammatory disorders and that administration of the sodium channel blocker flecainide in the EAN model, significantly increased the number of functional axons and significantly decreased axonal loss.
Physical trauma (car accident, gunshot, falls, etc.) or disease (polio, spina bifida, Friedreich's Ataxia, etc.) can lead to spinal cord injury (SCI)—damage to the spinal cord that results in a loss of function such as mobility or feeling. The spinal cord does not have to be severed in order for a loss of functioning to occur. In fact, in most people with SCI, the spinal cord is intact, but the damage to it results in loss of function. The extent of loss of function will vary depending on the area of injury but can range from quadriplegia, partial loss of function or dexterity in the arms and hands, paraplegia, poor trunk control as the result of lack of abdominal muscle control and reduced control of the hip flexors and legs. Besides a loss of sensation or motor functioning, individuals with SCI also experience other changes. For example, they may experience dysfunction of the bowel and bladder. Very high injuries (C-1, C-2) can result in a loss of many involuntary functions including the ability to breathe, necessitating breathing aids such as mechanical ventilators or diaphragmatic pacemakers. Other effects of SCI may include low blood pressure, inability to regulate blood pressure effectively, reduced control of body temperature, inability to sweat below the level of injury, and chronic pain.
Secondary cell injury due to spinal cord trauma results, in part, from the accumulation of calcium ions within injured neurons and their axons. As noted above, this arises due to reverse operation of the sodium-calcium exchanger, which in turn is triggered by an increase in intracellular sodium concentration via persistently activated voltage-gated sodium channels. Pharmacological blockade of sodium channels has been shown to prevent axonal degeneration and preserve function after injury to central nervous system white matter tracts.
Many peripheral or central neuropathic conditions commonly result in pain. Pain can be classed as acute (or nociceptive) or neuropathic.
Nociceptive pain is mediated by thermal, mechanical, electrical or chemical stimulation of pain receptors, known as nociceptors, which are located in skin, bone, connective tissue, muscle and viscera. Its purpose is to serve as a warning of potential ongoing tissue damage and is experienced in and around the point of injury. It usually responds to opioid and/or NSAID treatment. In the main, as healing progresses, the pain and inflammation associated with an injury abates and resolves.
In contrast, individuals may experience pain in the absence of an obvious tissue injury, or suffer (either continuously or periodically) chronic or protracted pain long after the injured tissue is apparently healed. Such pain serves no protective function and is predominantly neuropathic in nature, thus referred to as neuropathic pain, or chronic (nerve) pain. Neuropathic pain has been variously described as pain that results from a pathologic change in nerves or pain initiated or caused by a primary lesion or dysfunction in the nervous system (Mersky and Bogduk, Classifications of Chronic Pain, 2nd Ed., Seattle IASP Press: 1994, 394; De Andres and Garcia-Ribas, Pain Practice, 2003, 3:1-7) and can be described as burning, electric, tingling and shooting in nature. Neuropathic pain is associated with a variety of disease states and presents in the clinic with a wide range of symptoms (Woolf and Mannion, Lancet, 1999, 353:1959-64). The damage to the nerves may be caused by accidental or surgical injury, by metabolic disturbances such as diabetes or vitamin B12 or other nutrient deficiency, by ischaemia, by radiation, by autoimmune attack, by cytotoxic drugs used in cancer chemotherapy by alcohol, by infections, especially viral infections, particularly with the herpes virus, by tumours, by degenerative diseases, or by unknown factors such as may be operative in trigeminal and other neuralgias. Neuropathic pain does not require specific pain receptor stimulation although such stimulation can add to the intensity of the pain sensation.
Neuropathic pain is often characterised by chronic allodynia and/or hyperalgesia. Allodynia is pain resulting from a non-noxious stimulus, i.e. a stimulus that does not ordinarily cause a painful response, e.g. a light touch. Hyperalgesia, on the other hand, is an increased sensitivity to noxious stimuli (injury), i.e. a greater than normal pain response, and can be further defined as primary, occurring immediately in the vicinity of an injury, or secondary, occurring in undamaged area remote from an injury. Neuropathic pain is usually unresponsive to treatments used for nociceptive pain.
It is estimated that neuropathic pain affects over 26 million people worldwide and despite its common occurrence, remains one of the most poorly understood and untreated conditions in primary care, and can have a debilitating effect on almost all aspects of a sufferer's life. It has been associated with depression, anxiety, loss of independence and can impact on an individual's relationships and ability to work. The annual cost of neuropathic pain in the United States alone, including medical expenses, lost income and lost productivity is estimated to be $100 billion. The condition is particularly prevalent amongst the elderly and is experienced by a significant proportion of patients suffering from other disease states such as diabetes and advanced cancer.
Sodium channel blockers have been reported as potentially useful agents in the treatment of neuropathic pain (Tanelian et al, Pain Forum, 1995, 4(22):75-80; Kyle and Ilyin, J. Med. Chem., 50: 2583-8, 2007; Ilyin, et al, J. Pharmacol. Exper. Ther., 2006, 318:1083-93). There is evidence that sodium channel blockers selectively suppress etopic neural firing in injured (unmyelinated) nerves, which have an accumulation of sodium channels, and studies carried out on known blockers, such as carbamazepine, phenytoin, lidocaine and mexiletine, have demonstrated utility in the treatment of various types of neuropathic pain. Consistent with this, is the demonstration that sodium channels accumulate in the peripheral nerve sites of axonal injury and also in second order sensory neurons in pain pathways in the spinal cord. Alterations in the either the level of expression or distribution of sodium channels within an injured nerve, therefore, have a major influence on the pathophysiology of pain associated with this type of trauma.
Epilepsy is a disorder arising from abnormal or excessive bursts of electrical activity (firing) in the brain, which manifests as recurring seizures over a period of time. A person can develop epilepsy at any age. While many sufferers will experience at least one seizure before adulthood, a rapidly growing demographic is the over 55 years population, prone to cerebrovascular, respiratory and cardiac events that can lead to seizures. While the recurrence of seizures is conveniently described by a single term, the disorder can be further categorised by the seizure types of varying aetiologies, such as infections (e.g. meningitis and encephalitis), lack of oxygen, brain injury, brain tumours and neurodegenerative diseases. Susceptibility to or development of epilepsy may also be due to genetic factors, particularly in children. Mutations in genes encoding neuronal voltage-gated sodium channels are the most common known genetic cause of epilepsy, and are implicated in severe myoclonic epilepsy of infancy (SMEI), generalized epilepsy with febrile seizures plus (GEFS+), simple febrile seizures, benign familial infantile seizures (BFIS), and benign familial neonatal-infantile seizures (BFNIS).
Seizures typically fall into two categories: partial or focal seizures, and generalised seizures.
Partial or focal seizures commence in a focal point of the brain and therefore affect the part of the body that is controlled by that part of the brain. These types of seizures can be the result of tumours, stroke or head injury.
Partial or focal seizures may then be further categorised. Simple partial seizures affect only one part of the brain and the symptoms are dependent upon the corresponding brain function. The sufferer usually remains alert during the seizure, which is of relatively short duration, and may involve involuntary movement or spasm of limbs, sensory disturbances, feelings of déjà vu or feelings of nausea. Complex partial seizures start in a small area of the temporal or frontal lobes and cause an impairment of awareness or responsiveness, in which the conscious state is altered, rather than lost. Memory loss is sometimes associated with complex partial seizures.
Primary generalised seizures involve the whole brain and thus affect the whole body. Genetic factors typically govern these types of seizures. Primary generalised seizures produce loss of consciousness either briefly or for a longer period of time, and are further sub-categorized into several major types, some convulsive and some non-convulsive.
Absence seizures (petit mal seizures) are non-convulsive. The seizure manifests as starring or fluttering of eyelids and usually lasts only several seconds. Atonic seizures affect muscle tone causing the person to collapse. Myoclonic seizures are brief shock-like jerks of muscles. Tonic seizures result in sudden stiffening movements. Tonic-clonic seizures (grand mal seizures) may last several minutes. The subject's body stiffens and falls, and their limbs jerk in rhythmic movements.
Secondary generalised seizures occur when a disturbance occurs in a focal part of the brain (partial seizure) but then spreads throughout the brain.
In humans, Nav1.2 mutations are associated with inherited epilepsy (Heron, et al, Lancet, 2002, 360:851-2) and other forms of epilepsy, such as generalised epilepsy with febrile seizures (Sugawara, et al, Proc Natl Acad Sci USA, 2001, 98:6384-9)
Studies have shown that protection from seizures can be achieved by inhibition of sodium channels, in particular Nav1.1 and Nav1.2 channels. For example, blocking high-frequency repetitive spike firing, believed to occur during the spread of seizure activity helps protect against generalized tonic clonic and partial seizures. (Rogawski and Loscher, Nature Reviews, 2004, 5:553-64, and references therein).
Current therapies involve anticonvulsant drugs, also known as anti-epileptic drugs (AEDs), which modify the bursting properties of neurons and reduce synchronization in localized neuronal ensembles, as well as inhibiting the spread of abnormal firing to distant sites. Nevertheless, a proportion of patients continue to experience seizures, and/or suffer from unwanted side effects.
Abnormalities of neuronal excitability are also implicated in migrane, and sodium channel blockers have been postulated as having potential use in its treatment and/or prevention, with controlled clinical trials suggesting that certain Nav blocker anti-eplilepic drugs prevent migrane (Mantegazza, M., et al, Lancet Neurol 2010, 9:413-424, and references cited therein).
Sodium channels, particularly Nav1.1, Nav1.5 and Nav1.6 are also up-regulated in models of autoimmune and inflammatory disorders and thus may be implicated in such disorders.
In recent years there has been increasing evidence correlating the function of ion channels with cancer progression, and sodium channel activity has been clearly associated with invasion and metastasis behaviours of several types of cancer, including breast, colon, lung, ovary and prostate. Prostate cancer cells have been reported to overexpress Nav1.7 subunits, whereas Nav1.5 is overexpressed in breast, colon and ovary cancer cells. (Hermandez-Plata, E., et al, International J Cancer, 2012, 130: 2013-2023, and the references therein). It has also been observed that voltage-gated sodium channels are up-regulated in strongly metastatic cancers and that inhibiting these channels suppresses tumour cell motility and invasiveness in breast, prostate, lung and cervical cancers (Onkal, R., et al, European J Pharm, 2009, 625: 206-219. Hermandez-Plata, E., et al (International J Cancer, 2012, 130: 2013-2023) also demonstrated that a selective Nav1.6 inhibitor reduced in vitro invasiveness of primary cultures from human cervical cancers.
An increasing body of evidence correlates abnormal expression and function of sodium channels with disorders such as neuropathic pain, multiple sclerosis, epilepsy, migraine and cancer, and data already indicates that sodium channel blockers are efficacious for a range of diseases and disorders (Mantegazza, M., et al, supra). Given the individual and social impact of these diseases and disorders and other conditions in which excessive, undesirable or otherwise unwanted sodium channel activity is involved or implicated, there remains the need for new therapies that may ameliorate, relieve, prevent or otherwise improve one or more of their symptoms, or the conditions themselves.
Thus, compounds that can inhibit or otherwise modulate undesirable activity, such as excessive firing, of one or more voltage-gated sodium channel sub-types may be useful in the treatment and/or prevention of neuropathies or diseases associated with such activity.